Filter Mating Research Articles

Phage ϕC2 Mediates Transduction of Tn 6215 , Encoding Erythromycin Resistance, between Clostridium difficile Strains

ABSTRACTIn this work, we show that Clostridium difficile phage ϕC2 transduces erm(B), which confers erythromycin resistance, from a donor to a recipient strain at a frequency of 10−6 per PFU. The transductants were lysogenic for ϕC2 and contained the erm(B) gene in a novel transposon, Tn6215. This element is 13,008 bp in length and contains 17 putative open reading frames (ORFs). It could also be transferred at a lower frequency by filter mating.

PrgK, a Multidomain Peptidoglycan Hydrolase, Is Essential for Conjugative Transfer of the Pheromone-Responsive Plasmid pCF10

Peptidoglycan (PG) hydrolases associated with bacterial type IV secretion systems (T4SSs) are thought to generate localized lesions in the PG layer to facilitate assembly of the translocation channel. The pheromone-responsive plasmid pCF10 of Enterococcus faecalis encodes a putative cell wall hydrolase, PrgK, and here we report that a prgK deletion abolished functionality of the pCF10-encoded T4SS as monitored by pCF10 conjugative transfer. Expression in trans of wild-type prgK fully complemented this mutation. PrgK has three potential hydrolase motifs resembling staphylococcal LytM, soluble lytic transglycosylase (SLT), and cysteine-, histidine-dependent amidohydrolase/peptidase (CHAP) domains. Complementation analyses with mutant alleles established that PrgK bearing two hydrolase domains in any combination supported near-wild-type plasmid transfer, and PrgK bearing a single hydrolase domain supported at least a low level of transfer in filter matings. When exported to the Escherichia coli periplasm, each domain disrupted cell growth, and combinations of domains additionally induced cell rounding and blebbing and conferred enhanced sensitivity to osmotic shock. Each domain bound PG in vitro, but only the SLT domain exhibited detectable hydrolase activity, as shown by zymographic analyses and release of fluorescent PG fragments. Genes encoding three T4SS-associated, putative hydrolases, Lactococcus lactis CsiA, Tn925 Orf14, and pIP501 TraG, partially complemented the ΔprgK mutation. Our findings establish that PrgK is an essential component of the pCF10-encoded Prg/Pcf T4SS and that its hydrolase domains coordinate their activities for full PrgK function. PrgK is indispensable for plasmid transfer in liquid matings, suggestive of a role in formation or stabilization of mating junctions.

Single-Cell Analyses Revealed Transfer Ranges of IncP-1, IncP-7, and IncP-9 Plasmids in a Soil Bacterial Community

The conjugative transfer ranges of three different plasmids of the incompatibility groups IncP-1 (pBP136), IncP-7 (pCAR1), and IncP-9 (NAH7) were investigated in soil bacterial communities by culture-dependent and culture-independent methods. Pseudomonas putida, a donor of each plasmid, was mated with soil bacteria, and green fluorescent protein (GFP), encoded on the plasmid, was used as a reporter protein for successful transfer. GFP-expressing transconjugants were detected and separated at the single-cell level by flow cytometry. Each cell was then analyzed by PCR and sequencing of its 16S rRNA gene following either whole-genome amplification or cultivation. A large number of bacteria within the phylum Proteobacteria was identified as transconjugants for pBP136 by both culture-dependent and culture-independent methods. Transconjugants belonging to the phyla Actinobacteria, Bacteroidetes, and Firmicutes were detected only by the culture-independent method. Members of the genus Pseudomonas (class Gammaproteobacteria) were identified as major transconjugants of pCAR1 and NAH7 by both methods, whereas Delftia species (class Betaproteobacteria) were detected only by the culture-independent method. The transconjugants represented a minority of the soil bacteria. Although pCAR1-containing Delftia strains could not be cultivated after a one-to-one filter mating assay between the donor and cultivable Delftia strains as recipients, fluorescence in situ hybridization detected pCAR1-containing Delftia cells, suggesting that Delftia was a "transient" host of pCAR1.

Genetic analysis of Tn916-like elements conferring tetracycline resistance in clinical isolates of Clostridium difficile

As an important clinically relevant pathogen, Clostridium difficile has a high multidrug resistance rate. Conjugative transposons play a vital role in its resistance phenotype. In the present study, 34 tetracycline-resistant clinical isolates of C. difficile were studied to detect tetracycline resistance genes and the presence of transposons. Thirty-two isolates were found to harbour Tn916-like elements carrying the tet(M) resistance gene, of which only one copy existed in the genome by Southern blot analysis. To characterise the genetic organisation of the Tn916-like elements, overlap PCR assays were performed with nine primer pairs, revealing three types of elements designated T1 to T3. The prevalent element T1 lacking PCRA (ORF23 to ORF21) and PCRB (ORF21 to ORF20) products, present in the epidemic ST37 clone, was further analysed by genome walking PCR in the left and right end sequences of the novel Tn916-like element. A gene coding for an FtsK/SpoIIIE family protein was found to replace the ORF24 to ORF21 region in Tn916. Moreover, the element could hardly conjugate between cells by filter mating experiments. These findings suggest that the dissemination of Tn916-like elements in epidemic ST37 strains in China was likely to have been conferred by clonal spread, signifying the importance of future surveillance and characterisation of conjugative transposons.

Interference in Pheromone-Responsive Conjugation of a High-Level Bacitracin Resistant Enterococcus faecalis Plasmid of Poultry Origin

The current study reports on contact interference of a high-level bacitracin- resistant pheromone-responsive plasmid of Enterococcus faecalis strain 543 of poultry origin during conjugative transfer of bcr antimicrobial resistance genes using a polyclonal antiserum aggregation substance44–560 (AS). After induction with pheromones produced by the recipient strain E. faecalis JH2-2, clumping of the donor E. faecalis strain 543 was observed as well as high transfer frequencies of bcr in short time broth mating. Filter mating assays from donor strain E. faecalis 543 to the recipient strain E. faecalis JH2-2 revealed conjugative transfer of asa1 (AS), bcrRAB and traB (negative regulator pheromone response) genes. The presence of these genes in transconjugants was confirmed by antimicrobial susceptibility testing, PCR, Southern hybridization and sequencing. A significant reduction in formation of aggregates was observed when the polyclonal anti-AS44–560 was added in the pheromone-responsive conjugation experiments as compared to the induced state. Moreover, interference of anti-AS44–560 antibodies in pheromone-responsive conjugation was demonstrated by a reduction in horizontal transfer of asa1 and bcr genes between E. faecalis strain 543 and E. faecalis JH2-2. Reducing the pheromone-responsive conjugation of E. faecalis is of interest because of its clinical importance in the horizontal transfer of antimicrobial resistance.

Detection of VIM-34, a novel VIM-1 variant identified in the intercontinental ST15 Klebsiella pneumoniae clone

Sir, Enterobacteriaceae producing metallo-b-lactamases (MBLs), and particularly VIM-type MBLs, have frequently been implicated in hospital outbreaks across Europe, blaVIM genes having been linked to Tn402 derivatives, epidemic plasmids (IncN, IncI1, IncHI2) and occasionally with particular Enterobacteriaceae clones. VIM enzymes have been classified in three clusters (VIM-1, VIM-2 and VIM-7) according to their amino acid sequences (http://www. lahey.org/studies), VIM-1 and VIM-2 being the most widespread variants. In this study, we report the molecular epidemiology and the antibiotic susceptibility profiles of Klebsiella pneumoniae clinical isolates producing VIM-34, a novel VIM-1 variant identified in Portugal. In October 2011 and October 2012, two K. pneumoniae isolates (strains K43 and K47, respectively) showing reduced susceptibility to carbapenems (MICs 0.38–1.0 mg/L) were recovered from urine samples of hospitalized patients in a general hospital in northern Portugal (Hospital Pedro Hispano). They are the only carbapenemase-producing Enterobacteriaceae isolates identified in this hospital since the beginning of 2011, when reference protocols for carbapenemase detection were adopted. Antimicrobial susceptibility tests were performed using the Etest for b-lactams and disc diffusion for all other antimicrobial agents. These showed that all isolates were resistant to diverse cephalosporins, aztreonam,b-lactam/b-lactamase inhibitor combinations (Table 1), nalidixic acid, ciprofloxacin, chloramphenicol, gentamicin, kanamycin, netilmicin, streptomycin, tobramycin and sulphonamides, but susceptible to trimethoprim and amikacin (http://www.eucast.org/). Standard disc diffusion phenotypic tests using different b-lactams and b-lactamase inhibitors (cefotaxime, ceftazidime, imipenem; 0.2 mM EDTA, clavulanic acid), isoelectric focusing, PCR and sequencing demonstrated the production of VIM-34 (pI1⁄45.4) (GenBank accession number JX013656), a novel VIM-type enzyme differing from VIM-1 by one amino acid change (V113I, according to MBL standard numbering scheme) and co-production of SHV-1 (pI1⁄47.6) and SHV-12 (pI1⁄48.2) extended-spectrum b-lactamase. We could not identify the origin of these isolates but as both patients had multiple previous hospitalizations (including in other hospitals) and carried the same novel blaVIM type, a common nosocomial source seems more plausible than community acquisition. The blaVIM-34 from the K47 isolate was cloned in the pBGS18 (kanamycinresistance)plasmidusingprimersVIM-EcoRI(5′-GGGAATT CGCAGTCGCCCTAAAACAAAG-3′) and VIM-PstI (5′-AACTGCAGCCGCTCCA ACGATTTGTTAT-3′) (restrictionsitesareunderlined),andtheexpression vector (pBGS18/VIM-34) was further introduced into Escherichia coli DH5a, as previously reported. MICs of different b-lactams were determined using the Etest (in triplicate) and compared with those corresponding to a blaVIM-1-carrying clone obtained in the same conditions (Table 1). The VIM-34-producing E. coli recombinant yielded b-lactam MIC values similar to those observed in the VIM-1-encoding transformant (with the exception of cefoxitin; Table 1). Because our experiments were performed in an isogenic context and identical standard experimental conditions, we are able to hypothesize that the substitution V113I has a low influence on the MICs of carbapenems, although further studies of enzymatic activity are required to confirm this observation. The isolates exhibited identical XbaI-PFGE profiles and clonal identification by multilocus sequence typing(http://www.pasteur.fr/ recherche/genopole/PF8/mlst/Kpneumoniae.html) revealed that they belong to the intercontinental ST15 K. pneumoniae clone, widely disseminated in different European countries and associated with the spread of extended-spectrum b-lactamases (CTX-M-15; diverse SHV types) and/or MBLs (VIM-1, NDM-1).Conjugation assays performed by broth and/or filter mating methods using E. coli HB101 (azide and kanamycin resistant, Lac-, plasmid free) as recipient at 228C and 378C (selection of transconjugants in MacConkey agar with 2 mg/L of ceftazidime and 130 mg/L of azide) failed to yield transconjugants either for blaVIM-34 or blaSHV-12. The location of bla (blaVIM-34, blaSHV-12) genes and plasmid characterization were accomplished by S1and I-CeuI-PFGE, and identification of incompatibility groups. In both isolates, blaVIM-34, blaSHV-12 and repHI2 probes hybridized in the same chromosomal band (I-CeuI-PFGE) whereas no signals were observed in the S1 gel, suggesting the acquisition of both bla genes by an IncHI2 plasmid and subsequent plasmid (whole or in part) integration. A chromosomal location for bla genes, including blaVIM, has been occasionally observed in different Enterobacteriaceae species. The linkage of blaVIM-34 to class 1 integrons and Tn402 derivatives was investigated by PCR (intI1, 5′CS-3′CS region, orf5, orf6, IS1326, IS1353, IS6100) and sequencing. blaVIM-34 was located within an 6 kb class 1 integron named In817 by INTEGRALL (http://integrall.bio.ua.pt/) (GenBank accession number JX185132), with an original array of gene cassettes comprising blaVIM-34, aacA4′, aphA15, aadA1b and catB2 (Figure S1; available

American crows as carriers of vancomycin‐resistant enterococci with vanA gene

We studied the vanA-carrying vancomycin-resistant enterococci (VRE) isolated from American crows in the United States during the winter 2011/2012. Faecal samples from crows were cultured selectively for VRE and characterized. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to examine epidemiological relationships of vanA-containing VRE. Isolates were tested in vitro for their ability to horizontally transfer the vancomycin resistance trait. VRE with the vanA gene were found in 15 (2.5%) of 590 crows samples, from which we obtained 22 different isolates. Enterococcal species were Enterococcus faecium (14) and E. faecalis (8). One, two and 19 isolates originated from Kansas, New York State and Massachusetts, respectively. Based on MLST analysis, E. faecium isolates were grouped as ST18 (6 isolates), ST555 (2), and novel types ST749 (1), ST750 (3), ST751 (1), ST752 (1). Enterococcus faecalis isolates belonged to ST6 (1), ST16 (3) and ST179 (4). All isolates were able to transfer the vancomycin resistance trait via filter mating with very high transfer range. Clinically important enterococci with the vanA gene occur in faeces of wild American crows throughout the United States. These migrating birds may contribute to the dissemination of VRE in environment over large distances. [Correction added after first online publication on 06 August 2013: The number of E. faecium ST752 isolate is now amended to '1', consistent with that shown in the 'Results' section and Figure 2.].

Identification of Tet45, a tetracycline efflux pump, from a poultry-litter-exposed soil isolate and persistence of tet(45) in the soil

To characterize a tetracycline resistance (Tc(R)) determinant, closely related to the TetL efflux pump, in a Bhargavaea cecembensis strain previously isolated from a poultry-litter-impacted soil. Genomic DNA of B. cecembensis DMV42A was shotgun cloned and expressed in Escherichia coli. Antimicrobial susceptibility testing and a [(3)H]tetracycline uptake assay were used to confirm the function of the target gene. Transferability of the gene was examined using filter matings and confirmed by PCR and sequencing. Real-time quantitative PCR was performed on soil metagenomic DNA to evaluate the prevalence of the gene in the soil from which B. cecembensis DMV42A was isolated and in more pristine local soils. The Tc(R) determinant from B. cecembensis DMV42A, designated Tet45, was identified as a tetracycline efflux pump sharing 78% amino acid identity with certain TetL proteins. In B. cecembensis DMV42A, tet(45) was adjacent to truncated and non-functional arsenic resistance genes with high sequence similarities to genes from staphylococcal plasmids. After filter matings, the tet(45) gene could be found in E. coli transconjugants, although the transfer mechanism was unknown. Tet45 homologues are also present in the genomes of several Bacillus cereus strains and a Bacillus thuringiensis strain. tet(45) was detected in the poultry-litter-impacted soil, and persisted at a similar level 2 years after removal of the chicken waste, although it was not detected in several more pristine soils. Tet45 is a tetracycline efflux pump closely related to TetL. Horizontal gene transfer may have contributed to the dissemination and persistence of tet(45) in a poultry-litter-impacted soil.

Conjugal transfer of bacteriocin plasmids from different genera of lactic acid bacteria into Enterococcus faecalis JH2-2

The lactic acid bacteria (LAB) are of great interest because of their food grade quality and industrial importance. In the recent past, the pediocin PA-1 like bacteriocin was found to be synthesized in cross-species and cross-genera. Hence, the present work was carried out in order to determine the transfer of plasmid encoded pediocin PA-1 like bacteriocin among LAB. The objective of this study is to demonstrate the dissemination of bacteriocin-encoded plasmid from Pediococcus acidilactici NCIM 5424, Enterococcus faecium NCIM 5423 and Lactobacillus plantarum Acr2 to Enterococcus faecalis JH2-2 under in vitro (filter mating method) and in situ (soymilk model) conditions. The fermentation of the soymilk was determined by the selected pediocin producers. E. faecium NCIM 5423 was able to transfer the bacteriocin only under in situ conditions, whereas the native pediocin producer P. acidilactici NCIM 5424 transferred the bacteriocin under both the methods used. The in situ method gave more transfer frequency, ranging from 10−7 to 10−4 transconjugants per recipient cell. No conjugal transfer by L. plantarum Acr2 was observed. The physiological conditions like pH and temperature were found to influence the production of bacteriocin in the obtained transconjugants. The results suggest the horizontal gene transfer (HGT) and the natural spread of pediocin PA-1-like bacteriocin among LAB present in their close vicinity by means of conjugation. The dissemination of pediocin PA-1-like bacteriocin under in situ conditions is noteworthy, and such bacteriocin producers can be useful in the fermentation of dairy products and construction of novel cultures.

English

Enterococcus faecalis has emerged as an important nosocomial pathogen worldwide, infections and outbreaks of Vancomycin-resistant enterococci (VRE) still appear to be rare in Baghdad. In the present study, 20 isolates of E. faecalis were collected from 252 clinical samples (151 urine and 101 blood) from different hospitals in Baghdad and 3 isolates from 50 stool samples from healthy people during the period 1/10/2010 to 1/12/2010. All isolates were identified through morphological, cultural and biochemical tests using Rapid ID-32 strep. Vancomycin-resistance phenotype was determined by the agar diffusion method; results showed that five clinical isolates (25%) and two faecal isolates (66.7%)were resistant to vancomycin. Minimum inhibitory concentrations (MIC) of clinical VRE isolates ranged between 4 to 512 µg/ml and the MICs for isolates of faecal origin were 64 and 128 µg/ml. Seven vancomycin-resistant E. faecalis isolates were examined for their drug resistance and plasmid DNAs. Of the 5 clinical isolates, 2 isolates exhibited resistance to azlocillin (Azl) and ofloxacillin (Ofx). All clinical and stool isolates were resistant to oxacillin (Ox), cefepime (Fep), rifampicin (Ra) and tetracycline (Te). Imipenem and ampicillin were found to be the most effective agents against the isolates. The detection of plasmid DNA by gel electrophoresis showed that some E. faecalis isolates carried a high molecular weight plasmid that was transferred to the recipient by filter mating. The putative presence of vanA gene was examined by PCR, using specific primers. Positive PCR amplifications were obtained in all 7 isolates for vanA gene.Vancomycin resistant E. faecalis increased gradually in Baghdad hospitals and high dissemination of vanA gene, which encoded high resistance level to vancomycin. Continued surveillance is required to prevent further spread of these serious resistances. Key words: Bacteria, vancomycin, Enterococcus faecalis

Tn6198, a novel transposon containing the trimethoprim resistance gene dfrG embedded into a Tn916 element in Listeria monocytogenes

To characterize Tn6198, a novel conjugative transposon from the clinical Listeria monocytogenes strain TTH-2007, which contains the tetracycline and trimethoprim resistance genes tet(M) and dfrG, respectively, and to assess its transferability in vitro and in situ. The complete sequence of Tn6198 was determined using a primer walking strategy. Horizontal gene transfer studies were performed by filter matings, as well as on the surface of smear-ripened cheese and smoked salmon. The presence of Tn916-like circular intermediates was determined by PCR. Antibiotic resistance was determined by the broth microdilution method and microarray hybridization. Sequencing of Tn6198 revealed that a 3.3 kb fragment containing dfrG was integrated between open reading frames 23 and 24 of Tn916. Furthermore, an additional copy of Tn916 was present in L. monocytogenes TTH-2007. Both elements were transferred simultaneously and separately in vitro to recipients L. monocytogenes 10403S and Enterococcus faecalis JH2-2 by conjugation, resulting in either tetracycline- and trimethoprim-resistant or solely tetracycline-resistant transconjugants. On the surface of cheese and salmon, only L. monocytogenes 10403S transconjugants were detected. This study reports the first Tn916-like element associated with a trimethoprim resistance gene, as well as the first fully characterized transposon conferring multidrug resistance in L. monocytogenes. This is of concern, as trimethoprim is administered to listeriosis patients with β-lactam allergy and as Tn6198 has a large potential for dissemination, indicated by both intra-species and inter-genus transfer.

Isolation of a Novel Antibiotic Resistance Plasmid DNA from Hospital Isolates of Pseudomonas aeruginosa

Emergence and dissemination of antibiotic resistance plasmids are major concern for hospital care system and increases the cost and decreases effectiveness of available antibiotics used in treatment of hospitalized patients. In this study two Pseudomonas aeruginosa, two Escherichia coli, and a Klebsiella pneumoniae were isolated from Intensive Care Unit (ICU) of a university hospital, in Kerman, Iran. K. pneumoniae exhibited resistance to all antibiotics routinely used in our hospital for treatment of patients except meropenem, while, the other isolates were sensitive to carbapenems and ciprofloxacin. Plasmid analysis of the selected isolates showed the presence of a single plasmid with molecular weight 65Kb in the P. aeruginosa isolate1. The plasmid was named as pKUM and belonged to incompatibility group -2 (IncP-2). Conjugation by filter mating revealed that resistance associated with gentamicin, kanamycin, cefotaxime, and ceftazidime phenotypes were transferred to E. coli ATCC25922 (Rif r ) recipient cells at the frequencies of 3.13 × 10-5 and 5.3 ×10-7 respectively. The results were further supported by curing and transformation experiments. MIC to cefepime was ≥30 μg/mL both in donor as well as the transconjugants while decreased to 0.5µg/ mL in cured derivative. The plasmid pKUM was quite stable (86%) in both donor cells and the recipient. From above results we concluded that resistances to third generation of cephalosporins, aminoglycosides and cefepime in P. aeruginosa isolate1 were indeed encoded by a conjugative plasmid. Acquisition of cefepime resistance through plasmid complicates the therapy of neutropenic patients in ICU and increases the cost and mortality of these patients.

Vancomycin‐resistant enterococci in rooks (Corvus frugilegus) wintering throughout Europe

This study's aims were to assess the prevalence of, and to characterize, vancomycin-resistant enterococci (VRE) from rooks (Corvus frugilegus) wintering in Europe during 2010/2011. Faeces samples were cultivated selectively for VRE and characterized. Pulsed-field gel electrophoresis and multilocus sequence typing (MLST) were used to examine epidemiologic relationships of vanA-containing VRE. The vanA-carrying VRE were tested in vitro for mobility of vancomycin resistance traits. VRE were found in 62 (6%) of 1073 rook samples. Enterococcal species diversity comprised Enterococcus gallinarum (48 isolates), followed by E. faecium (9) and E. faecalis (5). Eight VRE harboured the vanA and ermB genes. Seven vanA-carrying VRE originated from the Czech Republic and one from Germany. All vanA-carrying VRE were identified as E. faecium. Based on MLST analysis, six vanA-positive isolates were grouped as ST92 type, one isolate belonged to ST121, and the remaining one was described as a novel type ST671. Seven out of eight isolates were able to transfer the vancomycin resistance trait via filter mating with a transfer rate of 8.95 ± 3.25 × 10(-7) transconjugants per donor. In conclusion, wintering rooks in some European countries may disseminate clinically important enterococci and pose a risk for environmental contamination.

In vitro development and transfer of resistance to chlortetracycline in Bacillus subtilis

The present criteria and rules controlling the approval of the use of probiotics are limited to antibiotic resistance patterns and the presence of antibiotic resistance genes in bacteria. There is little information available in the literature regarding the risk of the usage of probiotics in the presence of antibiotic pressure. In this study we investigated the development and transfer of antibiotic resistance in Bacillus subtilis selected in vitro by chlortetracycline in a stepwise manner. Bacillus subtilis was exposed to increasing concentrations of chlortetracyclineto induce in vitro resistance to chlortetracycline, and the minimal inhibitory concentrations were determinedfor the mutants. Resistant B. subtilis were conjugated with Escherichia coli NK5449 and Enterococcus faecalis JH2-2 using the filter mating. Three B. subtilis tetracycline resistant mutants (namely, BS-1, BS-2, and BS-3) were derived in vitro. A tetracycline resistant gene, tet (K), was found in the plasmids of BS-1 and BS-2. Three conjugates (BS-1N, BS-2N, and BS-3N) were obtained when the resistant B. subtilis was conjugated with E. coli NK5449. The conjugation frequencies for the BS-1N, BS-2N, and BS-3N conjugates were 4.57×10(-7), 1.4×10(-7), and 1.3×10(-8), respectively. The tet(K) gene was found only in the plasmids of BS-1N. These results indicate that long-term use of probiotics under antibiotic selection pressure could cause antibiotic resistance, and the resistance gene could be transferred to other bacteria. The risk arising from the use of probiotics under antibiotic pressure should be considered in the criteria and rules for the safety assessment of probiotics.

Transfer of antibiotic resistance determinants between lactobacilli isolates from the gastrointestinal tract of chicken

The aim of this study was to assess the potential horizontal transfer of genetic traits for antibiotic resistance between lactobacilli isolated from the chicken gut, both in vitro and in vivo. Thirty-seven Lactobacillus spp. strains isolated from the gizzard, small and large intestines and caeca of free-range broiler chickens showed multi-drug resistance as assessed by disc diffusion assays. The minimum inhibitory concentration (MIC) for vancomycin, tetracycline, erythromycin and chloramphenicol was determined in De Man, Rogosa and Sharpe broth in a microplate assay. Almost all the lactobacilli isolates were resistant to vancomycin (except strains belonging to the Lactobacillus acidophilus group) and to tetracycline (MIC≥128 μg/ml). Only five strains were resistant to erythromycin, and six to chloramphenicol. The transfer rate in filter mating experiments performed using L. acidophilus strain 4M14E (EmR), Lactobacillus vaginalis strain 5M14E (CmR), Lactobacillus salivarius strain 5C14C (EmR), and the 4G14L and 3C14C strains of Lactobacillus reuteri (CmR) showed a frequency of approximately 1×104 cfu/ml of double-resistant transconjugants for the different combinations. The exception was the L. salivarius 5C14C (EmR) and L. vaginalis 5M14E (CmR) mating combination, which produced no transconjugants. In vivo experiments performed in gnotobiotic mice by mating L. acidophilus 4M14E (EmR) with L. reuteri 3C14C (CmR), L. reuteri 4G14L (CmR) or L. vaginalis 5M14E (CmR) resulted in transconjugants at 3.95±0.29, 3.16±0.33, and 4.55±1.52 log10 cfu/g of faeces, respectively. Taken together, these data suggest that genetic exchange may occur between native bacterial strains within the gastrointestinal tract of chickens, which might maintain a dynamic gene pool conferring antibiotic resistance upon indigenous microbiota components, even in the absence of the pathogens. This possibility must be taken into account as a complementary criterion when lactobacilli are screened for probiotic use.

Multiple-antibiotic resistance of Enterococcus faecalis and Enterococcus faecium from cecal contents in broiler chicken and turkey flocks slaughtered in Canada and plasmid colocalization of tetO and ermB genes.

This study was conducted to characterize the antimicrobial resistance determinants and investigate plasmid colocalization of tetracycline and macrolide genes in Enterococcus faecalis and Enterococcus faecium from broiler chicken and turkey flocks in Canada. A total of 387 E. faecalis and E. faecium isolates were recovered from poultry cecal contents from five processing plants. The percentages of resistant E. faecalis and E. faecium isolates, respectively, were 88.1 and 94% to bacitracin, 0 and 0.9% to chloramphenicol, 0.7 and 14.5% to ciprofloxacin, 72.6 and 80.3% to erythromycin, 3.7 and 41% to flavomycin, 9.6 and 4.3% (high-level resistance) to gentamicin, 25.2 and 17.1% (high-level resistance) to kanamycin, 100 and 94% to lincomycin, 0 and 0% to linezolid, 2.6 and 20.5% to nitrofurantoin, 3 and 27.4% to penicillin, 98.5 and 89.7% to quinupristin-dalfopristin, 7 and 12.8% to salinomycin, 46.7 and 38.5% (high-level resistance) to streptomycin, 95.6 and 89.7% to tetracycline, 73 and 75.2% to tylosin, and 0 and 0% to vancomycin. One predominant multidrug-resistant phenotypic pattern was identified in both E. faecalis and E. faecium (bacitracin, erythromycin, lincomycin, quinupristin-dalfopristin, tetracycline, and tylosin). These isolates were further examined by PCR and sequencing for the genes encoding their antimicrobial resistance. Various combinations of vatD, vatE, bcrR, bcrA, bcrB, bcrD, ermB, msrC, linB, tetM, and tetO genes were detected, and ermB, tetM, and bcrB were the most common antimicrobial resistance genes identified. For the first time, plasmid extraction and hybridization revealed colocalization of tetO and ermB genes on a ca. 11-kb plasmid in E. faecalis isolates, and filter mating experiments demonstrated its transferability. Results indicate that the intestinal enterococci of healthy poultry, which can contaminate poultry meat at slaughter, could be a reservoir for quinupristin-dalfopristin, bacitracin, tetracycline, and macrolide resistance genes.

Introduction of OXA-48-producing Enterobacteriaceae to Israeli hospitals by medical tourism

The carbapenemase OXA-48 has been reported from different Mediterranean countries. It is mostly encoded on a single plasmid in various Enterobacteriaceae species. We characterized the epidemiological and molecular features of OXA-48-producing Enterobacteriaceae (OPE) in Israel. Epidemiological investigation was conducted by the National Center for Infection Control. Genotyping was performed using multilocus sequence typing. The bla(OXA-48)-carrying plasmids were investigated using S1 endonuclease and restriction fragment length polymorphism (RFLP). Conjugation efficiency of the bla(OXA-48)-carrying plasmids was studied in a filter mating experiment. Since 2007, four OPE-infected patients were identified, all non-Israeli (two Palestinian, one Jordanian and one Georgian). Three had prior hospitalization; two in Jordan and one in Georgia. The bla(OXA-48) gene was detected in three Escherichia coli strains belonging to different clonal complexes, one Klebsiella oxytoca and one Klebsiella pneumoniae sequence type 101, as previously reported from Tunisia and Spain. In all isolates, the bla(OXA-48) gene was located inside Tn1999.2 and was carried on a 60 kb plasmid with an identical RFLP pattern. The plasmid was able to conjugate from Klebsiella spp. to E. coli, and had a conjugation efficiency up to ~10000 times higher than that of pKpQIL. OPE, introduced mainly by medical tourism, are an emerging threat to patients from affected Mediterranean countries. The bla(OXA-48)-carrying plasmid demonstrated remarkable conjugation efficiency, which is probably important in the success of its dissemination.

Selection Pressure Required for Long-Term Persistence of bla CMY-2 -Positive IncA/C Plasmids

Multidrug resistance blaCMY-2 plasmids that confer resistance to expanded-spectrum cephalosporins have been found in multiple bacterial species collected from different hosts worldwide. The widespread distribution of blaCMY-2 plasmids may be driven by antibiotic use that selects for the dissemination and persistence of these plasmids. Alternatively, these plasmids may persist and spread in bacterial populations in the absence of selection pressure if a balance exists among conjugative transfer, segregation loss during cell division, and fitness cost to the host. We conducted a series of experiments (both in vivo and in vitro) to study these mechanisms for three blaCMY-2 plasmids, peH4H, pAR060302, and pAM04528. Results of filter mating experiments showed that the conjugation efficiency of blaCMY-2 plasmids is variable, from <10(-7) for pAM04528 and peH4H to ∼10(-3) for pAR060302. Neither peH4H nor pAM04528 was transferred from Escherichia coli strain DH10B, but peH4H was apparently mobilized by the coresident trimethoprim resistance-encoding plasmid pTmpR. Competition studies showed that carriage of blaCMY-2 plasmids imposed a measurable fitness cost on the host bacteria both in vitro (0.095 to 0.25) and in vivo (dairy calf model). Long-term passage experiments in the absence of antibiotics demonstrated that plasmids with limited antibiotic resistance phenotypes arose, but eventually drug-sensitive, plasmid-free clones dominated the populations. Given that plasmid decay or loss is inevitable, we infer that some level of selection is required for the long-term persistence of blaCMY-2 plasmids in bacterial populations.

Phenotypic and genetic characterization of multidrug-resistant Salmonella Infantis strains isolated from broiler chicken meats in Turkey

AbstractTwenty Salmonella Infantis strains resistant against kanamycin, tetracycline, neomycin, spectinomycin, sulphonamide, nalidixic acid and trimethoprim were selected for this study out of 103 Salmonella strains isolated from broiler samples collected from several markets in the Bolu and Ankara regions of Turkey. The resistance genes aadA1, aphA1, sul1, tet(A), dfrA5/dfrA14 and gyrA were determined for these multidrug-resistant S. Infantis strains. S. Infantis strains contained a mega plasmid with the molecular size of 206 kb. The strains were divided into three groups according to the pulsed field gel electrophoresis patterns of XbaI digested chromosomal DNA. A Ser83→Tyr83 point mutation was found in the gyrA gene of all quinolone-resistant isolates. Filter mating experiments showed that 206 kb plasmid transferred nalidixic acid resistance associated with class I integrons.

Emergence of NDM-1-producing Acinetobacter baumannii in China

To investigate the prevalence of bla(NDM-1) in Gram-negative bacilli in China. A total of 11 298 clinical Gram-negative bacilli, covering Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, were collected for PCR-based surveillance of bla(NDM-1) from 57 hospitals representing 18 provinces in China. For bla(NDM-1)-positive isolates, antibiotic susceptibilities were assessed and molecular typing was performed using PFGE. The genetic location of bla(NDM-1) was determined by analysis of PFGE profiles of S1 nuclease-digested genomic DNA and Southern blot hybridization. Plasmid transfer to E. coli recipients was investigated using filter mating and electroporation. Four A. baumannii isolates with bla(NDM-1) were identified in four different provinces in China: no positive isolates were detected among E. coli, K. pneumoniae and P. aeruginosa. These bla(NDM-1)-positive A. baumannii were resistant to all carbapenems and cephalosporins, and three remained susceptible to fluoroquinolones, aminoglycosides and colistin. The four NDM-1-producing A. baumannii were clonally diverse and carried bla(NDM-1) on different plasmids. Plasmids carrying bla(NDM-1) were successfully transferred from three of the four isolates to E. coli recipients, although the transconjugants and transformants were prone to lose the transferred plasmids after passage in the absence of ampicillin selection. We describe the emergence of A. baumannii producing NDM-1 in China. Systemic surveillance network should be established for monitoring these resistant bacteria.